Dataset Information

Unlatching of the stem domains in the Staphylococcus aureus pore-forming leukocidin LukAB influences toxin oligomerization.

ABSTRACT: Staphylococcus aureus (S. aureus) is a serious global pathogen that causes a diverse range of invasive diseases. S. aureus utilizes a family of pore-forming toxins, known as bi-component leukocidins, to evade the host immune response and promote infection. Among these is LukAB (leukocidin A/leukocidin B), a toxin that assembles into an octameric β-barrel pore in the target cell membrane, resulting in host cell death. The established cellular receptor for LukAB is CD11b of the Mac-1 complex. Here, we show that hydrogen voltage-gated channel 1 is also required for the cytotoxicity of all major LukAB variants. We demonstrate that while each receptor is sufficient to recruit LukAB to the plasma membrane, both receptors are required for maximal lytic activity. Why LukAB requires two receptors, and how each of these receptors contributes to pore-formation remains unknown. To begin to resolve this, we performed an alanine scanning mutagenesis screen to identify mutations that allow LukAB to maintain cytotoxicity without CD11b. We discovered 30 mutations primarily localized in the stem domains of LukA and LukB that enable LukAB to exhibit full cytotoxicity in the absence of CD11b. Using crosslinking, electron microscopy, and hydroxyl radical protein footprinting, we show these mutations increase the solvent accessibility of the stem domain, priming LukAB for oligomerization. Together, our data support a model in which CD11b binding unlatches the membrane penetrating stem domains of LukAB, and this change in flexibility promotes toxin oligomerization.

SUBMITTER: Ilmain JK

PROVIDER: S-EPMC10665946 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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Unlatching of the stem domains in the Staphylococcus aureus pore-forming leukocidin LukAB influences toxin oligomerization.

Ilmain Juliana K JK Perelman Sofya S SS Panepinto Maria C MC Irnov Irnov I Coudray Nicolas N Samhadaneh Nora N Pironti Alejandro A Ueberheide Beatrix B Ekiert Damian C DC Bhabha Gira G Torres Victor J VJ

The Journal of biological chemistry 20231004 12

Staphylococcus aureus (S. aureus) is a serious global pathogen that causes a diverse range of invasive diseases. S. aureus utilizes a family of pore-forming toxins, known as bi-component leukocidins, to evade the host immune response and promote infection. Among these is LukAB (leukocidin A/leukocidin B), a toxin that assembles into an octameric β-barrel pore in the target cell membrane, resulting in host cell death. The established cellular receptor for LukAB is CD11b of the Mac-1 complex. Here ...[more]

PMID: 37802313

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Project description:Staphylococcus aureus (S. aureus) is a serious global pathogen that causes a diverse range of invasive diseases and is notorious for antibiotic resistance. S. aureus utilizes a family of pore-forming toxins, known as bi component leukocidins, to evade the host immune response and promote infection. Among these is LukAB (leukocidin A, leukocidin B), a toxin that is secreted as a soluble heterodimer and assembles into an octameric beta barrel pore that is embedded in the host cell membrane, resulting in death of the host cell. The established cellular receptor for LukAB is CD11b of the Mac1 complex. LukAB variants from S. aureus clonal complexes (CC) 30 and 45 were recently described to use the proton channel hydrogen voltage gated channel 1 (HVCN1) as a receptor. Here we show that HVCN1 is an essential receptor used by all LukAB variants representing the major S. aureus clonal complexes, including CC8 LukAB, which belongs to the most prevalent lineage responsible for skin and soft tissue infections in the United States. We demonstrate that while each receptor is sufficient to recruit CC8 LukAB to the plasma membrane of phagocytes, both receptors are required for maximal lytic activity. Why LukAB requires two receptors, and how each of these receptors contribute to pore-formation remains unknown. To begin to resolve this, we performed an alanine scanning mutagenesis screen to identify mutations that allow CC8 LukAB to bypass the requirement for CD11b. We discovered thirty mutations primarily localized in the stem domain of LukA and LukB that enabled LukAB to exhibit enhanced cytotoxicity in the absence of CD11b. Using crosslinking, electron microscopy, and hydroxyl radical protein footprinting experiments we show these mutations alter the solvent accessibility of the stem domain which prime LukAB for oligomerization. Together, our data allow us to introduce a role for CD11b beyond toxin recruitment to the target cell: we propose a model in which CD11b binding unlatches the membrane penetrating stem domains of LukAB, and this change in flexibility promotes toxin oligomerization, driving pore-formation. The mass spectrometric raw files for the hydroxyl radical footprinting experiment are included here.

Project description:The macrophage response to planktonic Staphylococcus aureus involves the induction of proinflammatory microbicidal activity. However, S. aureus biofilms can interfere with these responses in part by polarizing macrophages toward an anti-inflammatory profibrotic phenotype. Here we demonstrate that conditioned medium from mature S. aureus biofilms inhibited macrophage phagocytosis and induced cytotoxicity, suggesting the involvement of a secreted factor(s). Iterative testing found the active factor(s) to be proteinaceous and partially agr-dependent. Quantitative mass spectrometry identified alpha-toxin (Hla) and leukocidin AB (LukAB) as critical molecules secreted by S. aureus biofilms that inhibit murine macrophage phagocytosis and promote cytotoxicity. A role for Hla and LukAB was confirmed by using hla and lukAB mutants, and synergy between the two toxins was demonstrated with a lukAB hla double mutant and verified by complementation. Independent confirmation of the effects of Hla and LukAB on macrophage dysfunction was demonstrated by using an isogenic strain in which Hla was constitutively expressed, an Hla antibody to block toxin activity, and purified LukAB peptide. The importance of Hla and LukAB during S. aureus biofilm formation in vivo was assessed by using a murine orthopedic implant biofilm infection model in which the lukAB hla double mutant displayed significantly lower bacterial burdens and more macrophage infiltrates than each single mutant. Collectively, these findings reveal a critical synergistic role for Hla and LukAB in promoting macrophage dysfunction and facilitating S. aureus biofilm development in vivo. Staphylococcus aureus has a propensity to form multicellular communities known as biofilms. While growing in a biofilm, S. aureus displays increased tolerance to nutrient deprivation, antibiotic insult, and even host immune challenge. Previous studies have shown that S. aureus biofilms thwart host immunity in part by preventing macrophage phagocytosis. It remained unclear whether this was influenced solely by the considerable size of biofilms or whether molecules were also actively secreted to circumvent macrophage-mediated phagocytosis. This is the first report to demonstrate that S. aureus biofilms inhibit macrophage phagocytosis and induce macrophage death through the combined action of leukocidin AB and alpha-toxin. Loss of leukocidin AB and alpha-toxin expression resulted in enhanced S. aureus biofilm clearance in a mouse model of orthopedic implant infection, suggesting that these toxins could be targeted therapeutically to facilitate biofilm clearance in humans.

Dataset Information

Unlatching of the stem domains in the Staphylococcus aureus pore-forming leukocidin LukAB influences toxin oligomerization.

Publications

Unlatching of the stem domains in the Staphylococcus aureus pore-forming leukocidin LukAB influences toxin oligomerization.

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